CN114191848B - Method for cleaning agarose microspheres - Google Patents
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Abstract
The invention provides a method for cleaning agarose microspheres, which comprises the steps of standing and layering agarose microsphere emulsion obtained by an emulsification-cooling method, separating an upper oil phase to obtain a lower agarose microsphere emulsion layer; adding water into the agarose microsphere emulsion layer, standing for layering, separating to obtain an upper emulsion layer and a lower agarose microsphere aqueous phase layer, and separating from the lower agarose microsphere aqueous phase layer to obtain agarose microspheres; and (3) repeatedly adding water into the upper emulsion layer for layering separation, mixing the agarose microspheres obtained by each separation, and washing to obtain the high-purity agarose microspheres. The invention firstly stands the emulsion to separate most of oil phase, thus greatly reducing the volume of the emulsion layer to be cleaned, improving the maximum cleaning amount which can be realized at a single time and saving the operation cost of equipment; meanwhile, the volume of the emulsion layer is reduced, so that the using amount of cleaning fluid is reduced, the cleaning cost is further reduced, and the method is suitable for industrial production; the obtained microspheres have good physicochemical properties after the crosslinking reaction is finished.
Description
Technical Field
The invention relates to the technical field of solid-liquid separation, in particular to a method for cleaning agarose microspheres.
Background
The agarose microspheres have extremely high added value, are mainly used for separating and purifying macromolecular bioactive substances such as viruses, proteins, enzymes and the like, and are widely applied to the industries such as biomedicine, blood products, vaccines and the like. The agarose microspheres as a natural polysaccharide biological filler have the advantages of good biocompatibility, porosity, hydrophilicity, no charged groups and the like, and are widely applied to the field of separation chromatography. Meanwhile, the surfaces of the agarose microspheres contain a large number of hydroxyl groups, and the agarose microspheres are easy to chemically react with a plurality of groups, so that the surfaces of the agarose microspheres are easy to modify, and groups with different functions are bonded, so that the agarose microspheres are suitable for different separation objects and separation requirements.
At present, agarose microspheres are mainly prepared by an emulsification-cooling method, a spraying method, a micro-fluidic droplet generation method and an inorganic dispersant emulsification method. The emulsification-cooling method is the earliest method for preparing agarose microspheres, and mainly utilizes the characteristic that agarose is solidified when being cooled after being heated and dissolved to prepare the agarose microspheres. The method has simple process flow and high production efficiency, and is still the most main agarose microsphere preparation method up to now.
The prior art prepares agarose microspheres by a reversed-phase emulsion polymerization method, which mainly comprises the steps of heating and dissolving agarose in water to be used as a water phase, adding an emulsifier into an organic solvent, heating and stirring to be used as an oil phase, dripping the water phase into the oil phase to be emulsified into droplet microspheres, and then cooling and solidifying to obtain the agarose microspheres. However, the microspheres obtained by this method exist in water-in-oil emulsion, and it is necessary to separate, wash and collect pure agarose microspheres. Because the emulsion is water-in-oil emulsion, if the suction filtration is directly adopted, although the dosage of the subsequent washing liquid can be reduced, the filter paper is easy to block, and the microsphere structure can be damaged, so the emulsion is inconvenient to use. If centrifugal separation and cleaning are adopted, as shown in figure 1, the microspheres are separated into three layers after centrifugation, the upper layer of the microspheres is oil-containing emulsion, more oil phase is still adsorbed on the surfaces of the separated microspheres and needs to be cleaned and removed by an organic reagent, and the microsphere structure is easily damaged by centrifugal force, so that aggregation is caused, and the microspheres are inconvenient to use directly. Therefore, the mainstream separation and cleaning method at present is to directly wash the emulsion with water and/or ethanol alternately so as to gradually separate the water phase microspheres from the oil phase and clean the oil phase on the surface. Patent CN 102233254A shows that agarose is spray cooled and solidified into spheres, which are then washed sequentially with petroleum ether, acetone, ethanol and water. Patent CN 109225177A shows that the obtained microspheres are washed with mixed detergent of petroleum ether and isopropanol, and then washed with petroleum ether and water, respectively. The cleaning method of the microspheres uses a large amount of organic reagents in actual production, has high recovery cost in the using process and easily causes environmental pollution in the production process.
In view of the above, there is a need to design an improved method for washing agarose microspheres to solve the above problems.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for cleaning agarose microspheres. Firstly, the emulsion is kept stand to separate most of the oil phase, so that the volume of an emulsion layer to be cleaned is greatly reduced, the maximum cleaning amount which can be realized at a single time can be improved, and the operation cost of equipment is saved; meanwhile, the volume of the emulsion layer is reduced, so that the using amount of the cleaning solution is reduced, and the cleaning cost is further reduced.
In order to realize the purpose, the invention provides a method for cleaning agarose microspheres, which comprises the following steps:
s1, dispersing an agarose hot water solution into a hot oil phase, uniformly mixing, and then cooling and solidifying at a preset cooling rate to obtain an agarose microsphere emulsion;
s2, standing and layering the agarose microsphere emulsion obtained in the step S1, and separating an upper oil phase to obtain a lower agarose microsphere emulsion layer;
s3, adding water into the agarose microsphere emulsion layer obtained in the step S2, stirring for a preset time, standing for layering, separating to obtain an upper emulsion layer and a lower agarose microsphere aqueous phase layer, and separating from the lower agarose microsphere aqueous phase layer to obtain agarose microspheres;
and S4, carrying out separation operation of the step S3 on the emulsifying layer obtained in the step S3 for multiple times, mixing the agarose microspheres obtained by each separation, and washing to obtain the high-purity agarose microspheres.
As a further improvement of the invention, in the step S2, the standing and layering time is more than 6h.
As a further improvement of the invention, the standing and demixing time is 12-30h, and the volume of the upper oil phase is more than 1/3 of the volume of the agarose microsphere emulsion.
As a further improvement of the invention, in the step S3, the volume ratio of the added water to the agarose microsphere emulsion layer is (0.5-3): 1.
As a further improvement of the invention, the stirring preset time is 5-20min, the stirring speed is 50-200rpm, and the standing and layering time is 10-60min.
As a further improvement of the present invention, in step S4, the emulsion layer obtained in step S3 is subjected to the separation operation of step S3 for 3 to 5 times.
As a further improvement of the invention, in step S1, the concentration of the agarose hot aqueous solution is 0.02-0.1g/mL, and the volume ratio of the agarose hot aqueous solution to the hot oil phase is 1 (2-10).
As a further improvement of the invention, in step S1, the temperature of the agarose hot aqueous solution and the hot oil phase is 60-100 ℃, the cooling rate is 2-5 ℃/min, and the temperature is reduced to below 20 ℃.
As a further improvement of the present invention, in step S1, the oil phase is one or more of liquid paraffin, petroleum ether, cyclohexane and toluene.
As a further improvement of the invention, a surfactant Span 80 is also added into the oil phase.
The invention has the beneficial effects that:
1. according to the cleaning method of the agarose microspheres, the existing cleaning method of the agarose microspheres is optimized through improvement, firstly, the emulsion is kept stand, most of oil phase is separated out, and the volume of an emulsion layer to be cleaned is greatly reduced, so that the maximum cleaning amount which can be realized at a single time can be improved, and the operation cost of equipment is saved; meanwhile, the volume of the emulsion layer is reduced, so that the using amount of the cleaning solution is reduced, and the cleaning cost is further reduced.
2. Compared with the existing agarose microsphere cleaning method, the agarose microsphere cleaning method has the advantages that the standing and oil phase separation step is adopted, so that the oil phase in the emulsion layer is greatly reduced, and the microsphere cleaning process is easier; meanwhile, after the emulsion layer is stirred and layered by adding pure water, the microspheres exist in the water phase, and the cleaning operation is more facilitated.
3. According to the invention, the agarose microspheres are collected and cleaned by adding water by utilizing the hydrophilic property of agarose microsphere liquid drops, the cleaning process does not depend on an organic reagent, the agarose microspheres can be cleaned by using purified water, the hidden danger of environmental pollution caused by the organic reagent is reduced, and the method is safe and environment-friendly; the method has the advantages of simple cleaning process, high and stable yield of the obtained agarose microspheres, suitability for large-scale industrial production, and good physicochemical properties of the obtained microspheres after the crosslinking reaction is finished.
Drawings
FIG. 1 is a schematic representation of the emulsion separation layer structure of example 1 and comparative examples 3 and 4.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in detail below with reference to specific embodiments.
It should be noted that, in order to avoid obscuring the present invention with unnecessary details, only the structures and/or processing steps closely related to the scheme of the present invention are shown in the specific embodiments, and other details not closely related to the present invention are omitted.
In addition, it should be further noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
The cleaning method of the agarose microspheres provided by the invention comprises the following steps:
s1, dispersing the hot agarose aqueous solution into a hot oil phase, uniformly mixing, and then cooling and solidifying at a preset cooling rate to obtain the agarose microsphere emulsion.
In step S1, the concentration of the hot aqueous agarose solution is 0.02-0.1g/mL, and the volume ratio of the hot aqueous agarose solution to the hot oil phase is 1 (2-10). The temperature of the agarose hot aqueous solution and the hot oil phase is 60-100 ℃, the cooling rate is 2-5 ℃/min, and the temperature is reduced to below 20 ℃. The oil phase is one or more of liquid paraffin, petroleum ether, cyclohexane and toluene. The oil phase is also added with a surfactant Span 80.
In this step, the hot aqueous agarose solution is emulsified in the oil phase to obtain a water-in-oil emulsion, wherein the aqueous agarose solution is dispersed in the oil phase in the form of small droplets to form an infinite number of droplet units. When the temperature is reduced, agarose in the droplets is gradually separated out and solidified (the agarose is only dissolved in hot water), each droplet forms an agarose microsphere, water molecules exist on the surface and in internal pores of the microsphere, and the whole emulsion still exists in a water-in-oil mode.
S2, standing and layering the agarose microsphere emulsion obtained in the step S1, and separating an upper oil phase to obtain a lower agarose microsphere emulsion layer.
In step S2, the standing and layering time is more than 6h. The standing time is preferably 12-30h, and the volume of the upper oil phase is more than 1/3 of the volume of the agarose microsphere emulsion. Through standing separation, because the volume of an oil phase in the emulsion is far larger than that of a water phase, and because the density of the water phase is larger than that of the oil phase, after standing for a certain time, part of the oil phase floats on an upper layer, and because of the action of the tension of liquid drops of the water-in-oil emulsion, the lower layer liquid still exists in the form of a water-in-oil emulsion layer, but the content of the oil phase is obviously reduced.
And S3, adding water into the agarose microsphere emulsion layer obtained in the step S2, stirring for a preset time, standing for layering, separating to obtain an upper emulsion layer and a lower agarose microsphere aqueous phase layer, and separating to obtain agarose microspheres from the lower agarose microsphere aqueous phase layer.
The volume ratio of the added water to the agarose microsphere emulsion layer is (0.5-3): 1. Stirring for 5-20min at 50-200rpm for 10-60min.
In the step, after water is added into the emulsion layer, the content of water in the whole liquid is equal to or gradually greater than the content of the oil phase, and due to the hydrophilicity of agarose microsphere liquid drops, more water phases are gradually gathered around the microspheres in the stirring process, the oil phase is extruded out, and after standing again, the water phase layer with the lower layer only containing the agarose microspheres can be obtained, and the content of the oil phase is very low. And the upper emulsifying layer is water-in-oil emulsion still containing a small amount of agarose microspheres, and the residual agarose microspheres are gradually extracted by repeatedly adding water and separating in the step S4. Thus, the agarose microspheres with high yield and high purity can be obtained, and the agarose microspheres have uniform particle size distribution and good physicochemical properties.
And S4, carrying out water adding separation operation in the step S3 on the emulsifying layer obtained in the step S3 for multiple times, mixing the agarose microspheres obtained by each separation, and washing to obtain the high-purity agarose microspheres.
In step S4, the emulsion layer obtained in step S3 is subjected to the separation operation of step S3 for 3 to 5 times.
Example 1
S1: preparation of agarose microspheres: weighing 4.0g of agar sugar powder in 100mL of purified water, and heating to completely dissolve the agar sugar powder to obtain a water phase; weighing 12g Span 80 in 300mL liquid paraffin, stirring uniformly, controlling the temperature to be 60-80 ℃ and taking the mixture as an oil phase. Dripping the water phase into the oil phase, stirring at 60-80 ℃ for 30-60 min, cooling to below 20 ℃ at the speed of 2-5 ℃/min, standing and solidifying for 1h to form the microspheres.
S2: standing the emulsion after cooling and solidifying into balls for 16-24 h at normal temperature, wherein the upper layer of the emulsion after standing and layering is a clear oil phase, about 200mL, and the lower layer is an emulsion layer; sucking out the upper oil phase to obtain an emulsion layer containing microspheres.
S3: adding purified water with the same volume into the obtained emulsion layer, stirring at 150rpm for 10min, standing for 15 min to separate the water phase from the emulsion layer, wherein the upper layer is the emulsion layer, the lower layer is the water phase, and the microspheres are deposited at the bottom of the water phase; and (4) extracting and collecting the upper emulsion layer, and directly filtering and collecting the lower aqueous phase.
S4: and repeating the step S3 for 3 times, mixing the microspheres collected for 3 times, and cleaning the microspheres with purified water. The volume of the oil phase precipitated in step S1 as a function of the standing time is reported in Table 1. It can be seen that the volume of the upper oil phase gradually increases with the increase of the standing time, when the standing time reaches 16h, the volume of the oil phase basically does not increase greatly, and after 24h, the volume of the oil phase is 200mL, which is 2/3 of the original volume of the oil phase. It is shown that all the oil phases cannot be separated directly by standing due to the stability of the water-in-oil emulsion itself, and that too long standing time increases the manufacturing cost.
TABLE 1 oil phase volume as a function of standing time
| Time | 1h | 2h | 3h | 6h | 9h | 12h | 16h | 20h | 24h |
| Oil phase volume/mL | 30 | 50 | 70 | 130 | 160 | 180 | 195 | 198 | 200 |
Example 2
Example 2 only increases the number of repetitions of step S3 as compared to example 1. In embodiment 2, the number of times of repeating S3 in step S4 is 4, and the rest of the steps are the same as those in embodiment 1, and are not described herein again. The mass of microspheres collected after each performance of step S3 is reported in table 2.
TABLE 2 influence of the number of operations of step S3 on the quality of the microspheres obtained
| Number of operations | 1 | 2 | 3 | 4 | 5 |
| Mass per gram of microspheres | 79.64 | 10.81 | 1.37 | 0.52 | 0.03 |
| Total mass of microspheres/g | 79.64 | 90.45 | 91.82 | 92.34 | 92.37 |
| Accounts for the total mass percentage | 86.22％ | 97.92％ | 99.40％ | 99.97％ | —— |
As can be seen from table 2, the number of operations of step S3 is at the 4 th time, almost all microspheres have been obtained, i.e. when the number of times step S3 is repeated in step S4 is 3, all microspheres in the emulsion layer can already be separated. And repeating the step S3 at least twice to obtain the agarose microspheres with the concentration of 99%. The microspheres obtained by the first separation have the largest mass, which shows that most of the microspheres can be basically kept stand and dispersed in the water phase after water is added for the first time. Therefore, the agarose microspheres can be efficiently separated from the emulsion only by standing and adding water for separation, and the low-cost and low-pollution cleaning and recovery are realized.
Examples 3 to 6
Examples 3 to 6 are different from example 1 in that the amount of purified water added and the stirring rate in step S3 were changed, and the remaining steps are the same as example 1 and will not be described again. Specific parameters are shown in table 3.
TABLE 3 variation of parameters and resulting microsphere masses for examples 3-6
| Examples | Purified water and emulsion layer volumeRatio of | Stirring rate/rpm | Mass of microspheres/g |
| 3 | 0.5:1 | 150 | 70.73 |
| 4 | 3:1 | 150 | 92.17 |
| 5 | 1:1 | 50 | 91.35 |
| 6 | 1:1 | 200 | 91.78 |
Examples 3 and 4 compared to example 1, the volume ratio of the purified water added to the emulsion layer in step S3 was only changed. As can be seen from table 3, the volume ratio of the purified water to the emulsion layer affects the mass of the finally obtained microspheres, and when the volume ratio is small, the mass of the obtained microspheres decreases. This is probably because when the purification water volume that adds is few, stir the back of standing, the breakdown of emulsion ability of water to water-in-oil emulsion layer reduces for the agarose microballon that subsides in water phase is closer with the emulsion layer, and the in-process of taking out the emulsion layer has some microballons to get into the emulsion layer along with it, and then influences gained microballon quality. When the volume ratio is large, the quality of the obtained microspheres is hardly affected, but an excessively high volume ratio increases the amount of the cleaning solution and increases the production cost.
Examples 5, 6 comparative example 1, only the stirring rate after adding purified water in step S3 was changed. From table 3, it can be seen that the stirring rate does not greatly affect the total mass of the microspheres obtained. However, too slow a stirring rate can reduce the separation of microspheres in the emulsion layer; too fast a rate will cause the emulsion layer to mix with the water phase, making it difficult to separate and form a new emulsion layer.
Comparative example 1
Preparation of agarose microspheres: weighing 4.0g of agar sugar powder in 100mL of purified water, and heating to completely dissolve the agar sugar powder to obtain a water phase; weighing 12g of Span 80 into 300mL of liquid paraffin, uniformly stirring, and controlling the temperature to be 60-80 ℃ to serve as an oil phase. Dropping the water phase into the oil phase, stirring at 60-80 deg.c for 30-60 min, cooling at 2-5 deg.c/min to below 20 deg.c, and stilling for 1 hr to form microsphere.
And (3) alternately cleaning the obtained emulsion with ethanol and purified water with the same volume, performing suction filtration for 10 times, then floating the cleaning solution without oil phase, collecting the obtained agarose microspheres, and cleaning and drying the agarose microspheres to obtain 92.71g.
Comparative example 2
Preparation of agarose microspheres: weighing 4.0g of agar sugar powder in 100mL of purified water, and heating to completely dissolve the agar sugar powder to obtain a water phase; weighing 12g Span 80 in 300mL liquid paraffin, stirring uniformly, controlling the temperature to be 60-80 ℃ and taking the mixture as an oil phase. Dripping the water phase into the oil phase, stirring at 60-80 ℃ for 30-60 min, cooling to below 20 ℃ at the speed of 2-5 ℃/min, standing and solidifying for 1h to form the microspheres.
And washing the obtained emulsion for 6 times by using petroleum ether, acetone, ethanol and water with equal volumes in sequence, then floating the cleaning solution without oil phase, collecting the obtained agarose microspheres, and cleaning and drying the agarose microspheres to obtain 92.21g.
Comparative example 3
Preparation of agarose microspheres: weighing 4.0g of agar sugar powder in 100ml of purified water, and heating to completely dissolve to obtain water phase; weighing 12g Span 80 in 300ml liquid paraffin, stirring evenly and controlling the temperature to be 60-80 ℃ to be used as an oil phase. Dripping the water phase into the oil phase, stirring at 60-80 ℃ for 30-60 min, cooling to below 20 ℃ at the speed of 2-5 ℃/min, standing and solidifying for 1h to form the microspheres.
Centrifuging the obtained emulsion for 5min at 5000rpm, separating the supernatant and the middle layer emulsion from the lower layer microspheres, washing the microspheres with ethanol and water of the same volume for 8 times, collecting the agarose microspheres, washing and drying to obtain 83.64g.
Comparative example 4
Preparing agarose microspheres: weighing 4.0g of agar sugar powder in 100ml of purified water, and heating to completely dissolve to obtain water phase; weighing 12g Span 80 in 300ml liquid paraffin, stirring evenly and controlling the temperature to be 60-80 ℃ to be used as an oil phase. Dripping the water phase into the oil phase, stirring at 60-80 ℃ for 30-60 min, cooling to below 20 ℃ at the speed of 2-5 ℃/min, standing and solidifying for 1h to form the microspheres.
Centrifuging the obtained emulsion for 5min at 5000rpm, separating the upper clear oil phase and the middle milky emulsion from the lower microspheres, washing the microspheres for 4 times by using equal volumes of petroleum ether, acetone, ethanol and water in sequence, collecting the agarose microspheres obtained, washing and drying, and collecting 84.36g.
In comparative examples 3 and 4, compared with example 1, the reason that the quality of the obtained microspheres is low is probably because the microspheres are directly close to the emulsion after centrifugation, and certain loss exists in the separation process; and because the microspheres are contacted with the emulsion after separation, the cleaning process of the microspheres still has obstacles and needs to be cleaned by using an organic reagent. The schematic diagram after the final separation process is shown in figure 1.
Performance testing
The microspheres obtained in the examples and the comparative examples are all screened to obtain microspheres with the particle size of 100-300 mu m, and the microspheres are crosslinked by a conventional method: and (3) taking 20g of the drained agarose microspheres, adding 20mL of purified water and 2mL of epoxy chloropropane, stirring for 30min at room temperature, dropwise adding 6mL of 5mol/L NaOH solution, and completing dropwise adding within 60min. Heating to 60 ℃ for reaction for 90min, and carrying out suction filtration and washing to obtain the crosslinked agarose microspheres.
Selecting the crosslinked microspheres
The chromatography column of (1) plugging the column outlet, mixing the medium with the column outletPouring the mixed slurry of water into a chromatographic column, standing, controlling the height of the medium bed layer to be 10.00cm +/-0.20 cm, and filling water into the upper end of the column. Opening the inlet of the column, continuously introducing three-stage water with 10 column volumes into the column at the flow rate of 0.5mL/min, and carrying out the test after the bed layer is stable.
Connecting the chromatographic column with a medium-low pressure chromatographic system. During the measurement, a certain flow velocity v is set from zero x (mL/min), and after maintaining this flow rate for 5min, the column pressure p (MPa) at that time was recorded. The flow rate was increased further and the column pressure at the corresponding flow rate was measured. Until the pressure reached 0.10MPa, at which time the corresponding flow rate was recorded as v 0.1 . The maximum flow velocity calculation formula: f max ＝60v 0.1 /S
In the formula:
fmax-maximum flow rate in centimeters per hour (cm/h);
v 0.1 -volumetric flow rate in milliliters per minute (mL/min) at a pressure of 0.10 MPa;
s-the cross-sectional area of the column in square centimeters (cm) 2 )；
60-minutes to an hour conversion factor.
TABLE 4 highest flow rates of examples and comparative examples
Example 1 was the same experiment as example 2, so only the highest flow rate of the microspheres obtained in example 1 was measured. As can be seen from Table 4, the highest flow rates of the microspheres obtained in the examples are all closer and higher than those of the comparative examples, which indicates that the microspheres obtained in the examples have better performance and good porosity and permeability.
In conclusion, the yield and the maximum flow rate of the microspheres prepared by the cleaning method are higher than those of the microspheres prepared by the prior art. On the basis of maintaining the performance of the existing microspheres, the invention improves the cleaning method of the microspheres, reduces the use and discharge of organic reagents and saves the ball manufacturing cost; and the method is safe, environment-friendly, simple to operate and suitable for large-scale industrial production.
Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the spirit and scope of the present invention.
Claims (7)
1. The method for cleaning the agarose microspheres is characterized by comprising the following steps of
S1, dispersing an agarose hot water solution into a hot oil phase, uniformly mixing, and then cooling and solidifying at a preset cooling rate to obtain an agarose microsphere emulsion;
s2, standing and layering the agarose microsphere emulsion obtained in the step S1, and separating an upper oil phase to obtain a lower agarose microsphere emulsion layer;
s3, adding water into the agarose microsphere emulsion layer obtained in the step S2, stirring for a preset time, standing for layering, separating to obtain an upper emulsion layer and a lower agarose microsphere aqueous phase layer, and separating from the lower agarose microsphere aqueous phase layer to obtain agarose microspheres;
s4, carrying out separation operation of the step S3 on the emulsion layer obtained in the step S3 for multiple times, mixing the agarose microspheres obtained by each separation, and washing to obtain high-purity agarose microspheres;
in the step S2, the standing and layering time is 12-30h, and the volume of the upper oil phase is more than 1/3 of the volume of the agarose microsphere emulsion;
in the step S3, the preset stirring time is 5-20min, the stirring speed is 50-200rpm, and the standing layering time is 10-60min.
2. The method for washing agarose microspheres of claim 1, wherein in step S3, the volume ratio of the added water to the emulsified layer of agarose microspheres is (0.5-3) to 1.
3. The method for washing agarose microspheres of claim 1, wherein in step S4, the emulsion layer obtained in step S3 is subjected to the separation operation of step S3 for 3-5 times.
4. The method for washing agarose microspheres of claim 1, wherein in step S1, the concentration of the agarose hot aqueous solution is 0.02-0.1g/mL, and the volume ratio of the agarose hot aqueous solution to the hot oil phase is 1 (2-10).
5. The method for cleaning agarose microspheres of claim 1, wherein in step S1, the temperature of the hot aqueous agarose solution and the hot oil phase is 60-100 ℃, the cooling rate is 2-5 ℃/min, and the temperature is reduced to below 20 ℃.
6. The method for cleaning agarose microspheres of claim 1, wherein in step S1, the oil phase is one or more of liquid paraffin, petroleum ether, cyclohexane and toluene.
7. The method for cleaning the agarose microspheres of claim 6, wherein a surfactant Span 80 is further added into the oil phase.
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